Antimicrobial susceptibility of Acinetobacter clinical isolates and emerging antibiogram trends for nosocomial infection management

Introduction: The drug resistant Acinetobacter strains are important causes of nosocomial infections that are difficult to control and treat. This study aimed to determine the antimicrobial susceptibility patterns of Acinetobacter strains isolated from different clinical specimens obtained from patients belonging to different age groups. Methods: In total, 716 non-duplicate Acinetobacter isolates were collected from the infected patients admitted to tertiary-care hospitals at Lahore, Pakistan, over a period of 28 months. The Acinetobacter isolates were identified using API 20E, and antimicrobial susceptibility testing was performed and interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: The isolation rate of Acinetobacter was high from the respiratory specimens, followed by wound samples. Antibiotic susceptibility analyses of the isolates revealed that the resistance to cefotaxime and ceftazidime was the most common, in 710 (99.2%) specimens each, followed by the resistance to gentamicin in 670 (93.6%) isolates, and to imipenem in 651 (90.9%) isolates. However, almost all isolates were susceptible to tigecycline, colistin, and polymyxin B. Conclusions: The present study showed the alarming trends of resistance of Acinetobacter strains isolated from clinical specimens to the various classes of antimicrobials. The improvement of microbiological techniques for earlier and more accurate identification of bacteria is necessary for the selection of appropriate treatments.


INTRODUCTION
Acinetobacter is a genus of Gram-negative coccobacilli, which are non-motile, oxidase-negative, and catalase-positive, and occur in pairs under magnification (1) .Acinetobacter species are opportunistic pathogens predominantly found in immunocompromised patients.They are widespread in nature, and regarded as commensal microbes of human skin and respiratory tract, however, they may cause serious infections, such as endocarditis, urinary tract infections, pneumonia, wound infections, meningitis, and septicemia, especially in individuals with impaired host defenses (2) .Infections caused by Acinetobacter species are acquired due to hospitalization, mechanical ventilation, respiratory failure, inadequate treatment, previous infection, or antibiotic therapy and catheterization (3) .Higher colonization rates of the throat, and respiratory and digestive tracts are well documented in several previous outbreaks (4) (5) .
Acinetobacter species are becoming increasingly resistant to nearly all routinely prescribed antimicrobial agents, including aminoglycosides, fluoroquinolones, and broad-spectrum β-lactams.The majority of strains are resistant to cephalosporin class of antimicrobials, whereas the resistance to carbapenems is increasingly reported (6) .The antimicrobial susceptibility testing showed differences between Acinetobacter species, with Acinetobacter baumannii being the most resistant strains (7) (8) .
The aim of the study was to compare the isolation frequency of Acinetobacter species form different clinical specimens and the frequency of antibiotic resistance in different age groups.Antimicrobial susceptibility assays were performed using different antibiotics that are frequently recommended by the clinicians.

Hospital and clinical isolates
This study was conducted at the Microbiology Section, Chughtai Lab, Lahore, Pakistan, between January 2012 and

Isolation and identification of bacteria
The preliminary identification of bacteria was done by standard microbiological procedures (9) .The obtained Acinetobacter isolates were further sub-cultured on MacConkey agar and confirmed by API 20NE (Biomerieux, France), according to the manufacturer's instructions.

Antimicrobial susceptibility analyses
Acinetobacter isolates were tested for the susceptibility to ampicillin/sulbactam, cefepime, cefotaxime, ceftazidime, ceftriaxone, imipenem, meropenem, amikacin, gentamicin, tobramycin, doxycycline, tigecycline, ciprofloxacin, levofloxacin, sulfamethoxazole/trimethoprim, piperacillin/ tazobactam, colistin, and polymyxin B (300U each).These analyses were performed by standard disc diffusion technique and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (5) .The bacterial suspension of each sample was made and compared with 0.5McFarland turbidity standard.The cartridges containing antimicrobial susceptibility discs (Oxoid, UK) were stored at between 4°C and -20°C, and used after the incubation at room temperature.Mueller-Hinton agar plates were inoculated and incubated at 35°C for 18h, and the zones of inhibition were measured.Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853) were used as reference strains, according to the CLSI protocol.
Broth dilution method was used to determine the susceptibilities of the isolated strains to tigecycline and colistin.The Food and Drug Administration (FDA), USA, approved breakpoints for the agents against Enterobacteriaceae were used to determine tigecycline susceptibility in Acinetobacter isolates (resistance, ≥8mg/L; susceptibility, ≤2mg/L).Minimum inhibitory concentration (MIC) breakpoints for colistin were used according to the CLSI guidelines (resistance, ≥4mg/L; susceptibility, ≤2mg/L) (10) (11) .

Statistical analysis
Statistical analyses were performed using Microsoft Excel 2010 (Microsoft, USA).The descriptive analysis was performed by calculating frequencies of isolation of Acinetobacter and percentage resistance among isolates to various antimicrobial agents.

RESULTS
In total, 716 Acinetobacter isolates were obtained (Table 1), with the majority from the respiratory samples, followed by the isolates from surgical or burn wounds.

Prevalence of Acinetobacter resistance according to the age group
The prevalence of Acinetobacter strain resistance was shown to be higher in individuals aged between 21 and 50 (Figure 1).The frequency of Acinetobacter strains isolated alone or in the combination with other bacterial strains isolated from the clinical specimens are different, with 72% of Acinetobacter strains isolated alone (519 isolates), as presented in Table 2.

Antimicrobial susceptibility profile
Antibiotic resistance profile of Acinetobacter isolates was determined using 18 antibiotics, and this revealed that the isolated strains were most commonly resistant to cefotaxime and ceftazidime [for both of them, 710 (99.2%) isolates were shown to be resistant], followed by the strains resistant to gentamicin [670 (93.6%)], and imipenem [651(90.9%)](Table 3).However, most of the tested isolates were susceptible to tigecycline, colistin, and polymyxin B.

DISCUSSION
The aim of this study was to characterize Acinetobacter samples obtained from the infected patients and the antimicrobial susceptibility of these isolates to various antibiotics commonly used in clinical practice.In total, 716 Acinetobacter strains were isolated.The higher isolation rates of Acinetobacter from the respiratory samples are in agreement with the results reported previously in other countries (12) .
The antibiotic resistance data collected around the world demonstrated that the resistance rates of Acinetobacter species to imipenem ranged from 0-40% between 2000 and 2004 (16) .The prevalence of imipenem resistance in Acinetobacter species increased from zero in 1991 to 50% in 2001, as shown in a study conducted in a Spanish hospital (17) .The resistant rates to ampicillin/sulbactam, imipenem, and meropenem were 51.6%, 26.3%, and 29.6%, respectively, of Acinetobacter isolates from 30 European centers (18) .
Colistin or tigecycline remain the treatment options for the management of most of the cases of infections caused by multidrug resistant A. baumannii strains.We showed that, among the strains we isolated, only 0.7% and 0.1% were resistant to tigecycline and colistin, respectively.This is in accordance with the previous studies (16) (18) .However, the emergence of Acinetobacter resistance against tigecycline and colistin is reported with an increasing frequency (18) (19) .In a surveillance study in Europe, the resistance of A. baumannii against polymyxin B was shown to be 2.7% (18) .A surveillance study in Greece showed that 3% of Acinetobacter strains isolated from ICU patients were resistant to colistin (20) .Furthermore, tigecycline and colistin resistance rates of A. baumannii strains isolated in Germany were 6% and 2.8%, respectively (21) .A study in Turkey reported considerably higher tigecycline resistance rates (25%) of Acinetobacter strains (22) .
Acinetobacter infections are sometimes accompanied by the infections caused by other microorganisms, and here, we showed that these species are mainly Candida, Escherichia coli, methicillinresistant Staphylococcus aureus (MRSA), Proteus, Klebsiella, and Pseudomonas species (Table 2).These co-infections may increase patient mortality, highlighting the importance of choosing the appropriate antimicrobial therapy (23) .The analyses of antibiotic resistance patterns according to the age groups showed that the number of the strains resistant to most of the antimicrobial agents increased with age, except the strains resistant to co-trimoxazole and piperacillin/tazobactam (Figure 1).Some differences in the antibiotic resistance patterns between the urinary tract pathogens isolated from different age groups were observed, however, the rate of resistance to different antibiotics was higher in pediatrics patients compared with that in the middle aged and elderly patients (24) .Another study demonstrated the differences between the age groups in susceptibility patterns among E. coli urinary tract isolates for all tested antibiotics except co-trimoxazole (25) .A microarray-based study showed that the antibiotic resistance gene diversity is age-related and that these genes accumulate in the members of human gut microflora starting from infancy, and their interactions become gradually more complex with age (26) .To the best of our knowledge, the differences in the Acinetobacter susceptibility to different antimicrobial agents between different age groups have not been reported before.
Taken together, this study identified the differences in the antibiotic resistance of Acinetobacter isolates obtained from hospitalized patients in Pakistan.Acinetobacter strains have the capacity to acquire antimicrobial resistance rapidly, and therefore, the resistance to even newer antimicrobials is reported worldwide.This allows them to cause nosocomial outbreaks in hospitals.Therefore, decreasing the pace of the emergence of antimicrobial resistance of Acinetobacter species is crucial, through the restricted use of antimicrobials, and the enforcement and surveillance of antibiotic Stewardship Programs in health care settings.

FIGURE 1 -
FIGURE 1 -Resistance patterns of Acinetobacter isolates to various classes of antimicrobial agents according to the different age groups.

TABLE 2 -Percentage of Acinetobacter isolated alone, or in combination with other isolates from clinical specimens.
MRSA: methicillin-resistant Staphylococcus aureus.

TABLE 3 -Resistance pattern of Acinetobacter isolates to various antimicrobial agents.
Sohail M et al. -Susceptibility of Acinetobacter clinical isolates